US4244940A - Single-incubation two-site immunoassay - Google Patents
Single-incubation two-site immunoassay Download PDFInfo
- Publication number
- US4244940A US4244940A US05/939,577 US93957778A US4244940A US 4244940 A US4244940 A US 4244940A US 93957778 A US93957778 A US 93957778A US 4244940 A US4244940 A US 4244940A
- Authority
- US
- United States
- Prior art keywords
- improved
- stimulating hormone
- labeled
- thyroid stimulating
- ligand
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/804—Radioisotope, e.g. radioimmunoassay
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S436/00—Chemistry: analytical and immunological testing
- Y10S436/807—Apparatus included in process claim, e.g. physical support structures
- Y10S436/808—Automated or kit
Definitions
- This invention relates to a novel method for a two-site immunoassay.
- Two-site immunoradiometric assays for antigens which can bind to at least two antibodies are known.
- Those known two-site immunoradiometric assays involve a first incubation of unlabeled antibody coupled to an insoluble matrix with the fluid containing the antigen having more than one antigenic determinant, followed by a second incubation with a labeled antibody. Woodhead, Addison and Hales, Br. Med. Bull., Vol. 39, No. 1, 44-49 (1974); Abraham (ed.), Handbook of Radioimmunoassay, Ch. 4 (Marcel Dekker, Inc., 1977).
- TSH human thyroid stimulating hormone
- Corning also markets an assay for the determination of human thyroxine-binding globulin ("TBG"). See Corning, TBG 125 I Radioimmunoassay Test System (December, 1977). In that assay, an immobilized antibody specific for TBG is added to a known amount of TBG standard or to a patient serum sample. After vortexing, radioactive 125 I labeled thyroxine (" 125 I T4") is added to the reaction mixture. According to Corning, the 125 I T4 will then partition between the binding sites on TBG and bovine serum albumin molecules present in the buffer. The TBG can thus become bound both to the immobilized antibody and to the 125 I T4.
- TBG thyroxine-binding globulin
- the solid phase antibody is mixed with the sample containing antigen (or hapten) to be determined and corresponding antigen (or hapten) which has been fluorescently labeled, so as to bind a quantity of labeled and unlabeled antigen.
- the solid phase is separated and the fluorescence measured by optical spectroscopy, the concentration of the unknown immune reactant being a function of the value of the fluorescence.
- This application also describes a two-site immunoassay method in which an excess of solid phase antibody is first reacted with the sample containing antigen (or hapten) to be determined, and the complex is subsequently reacted with an excess of fluorescently labeled antibody.
- the present invention in an improved two-site immunoassay method in which the sample containing the multivalent ligand to be determined, a labeled receptor for said ligand, and an unlabeled receptor for said ligand covalently bound to a solid-phase support (hereinafter referred to as "solid-phase receptor") are brought together in an aqueous medium to form a substantially stable suspension and in a single incubation mode or step to produce a two-phase system, wherein the solid phase contains the solid-phase receptor, a portion of which has become bound to ligand which in turn has become bound to a portion of the labeled receptor, and the liquid phase contains the unbound portion of the labeled receptor.
- the solid and liquid phases are separated and either phase analyzed for the labeled receptor, the concentration of which is a function of the concentration of ligand in the sample.
- a two-site immunoassay employing a single incubation mode or step provides a significant advantage over assay procedures involving more than one incubation by simplifying, shortening, and rendering more convenient the performance of the assay. This improvement in assay procedure is accomplished while maintaining acceptable assay characteristics such as precision, specificity, and stability, and in addition, is less subject to errors in timing, additions, and other manipulations.
- the novel two-site immunoassay of this invention can be applied to any ligand which can simultaneously become bound by two receptors.
- This group of ligands includes, but is not limited to placental, pituitary, calcium regulating, and adrenal medullary polypeptide hormones, protein and protein fragments; immunoglobulins (antibodies) of various classes; viral, bacterial, and protozoal organisms or particles; enzymes; and tumor-associated antigens.
- an assay for human thyroid stimulating hormone is described.
- This assay method utilizes a receptor for the ligand, which is any substance which binds the ligand with acceptable specificity and affinity. Accordingly, the receptor may be, for example, an antibody against the ligand. In a preferred example, antibodies are used as the receptor.
- the labeled receptor may be labeled with any of a number of known tracers, including, for example, radioactive tags, such as iodine 125 , or fluorescent labels.
- radioactively labeled antibodies are employed.
- an important component of this assay is the solid-phase support for unlabeled receptor which can form substantially stable suspensions in aqueous media, can be covalently bound to the unlabeled receptor, can be handled conveniently during manipulations such as pipetting and centrifuging, and exhibits low non-specific adsorption properties, or can be treated so that it exhibits such adsorption properties.
- the suspendability of the solid-phase support on which the unlabeled receptor is immobilized allows for the addition of an excess of solid-phase receptor, which binds the ligand in the sample, and yet permits further participation of the solid phase receptor-ligand complex in reactions with the labeled receptor.
- a suitable solid-phase support is derivatized polyacrylamide particles in a size range of 0.10 to 10 microns, which provides a substantially stable suspension during the single incubation period.
- a preferred embodiment of this two-site immunoassay having a single incubation mode provides high specificity for TSH by using highly purified antibodies.
- the hormones TSH, HCG, FSH, and LH are each composed of similar alpha and distinct beta polypeptide chains.
- the hormones FSH, LH, and HCG may interfere with the assay for TSH due to the cross reactivity of the antibodies to those hormones.
- Improved specificity for TSH of this assay is achieved by a two step purification procedure for the labeled antibody and, if needed, purification of the unlabeled antibody to obtain antibodies of high affinity for TSH and low affinity for other hormones, for example HCG.
- Prepurification of receptor by these means constitutes an approach in dealing with inherent cross-reactivity due to common antigenic determinants in various subclasses of ligands and constitutes an improvement over prior art.
- prepurification of antibody makes it possible to introduce 125 I into a higher proportion of specific antibody molecules (subclass of the IgG fraction), and thus more of the radioactive label is usable in the assay.
- the purification procedure for antibody to be labeled involves known principles of affinity chromatography to an immunoglobulin cut of rabbit anti-serum to TSH.
- the immunoglobulins are first passed over an HCG column to remove anti-alpha chain antibodies, and then over a TSH column.
- the specific anti-beta TSH antibodies bind to the TSH column, and they are eluted off in stages so that the final antibody for labeling consists of essentially pure anti-beta TSH antiboidies of high affinity.
- the unlabeled antibody may be evaluated for TSH specificity and, if necessary, either passed over an HCG column prior to coupling to the solid phase support or absorbed with HCG after coupling to the solid phase support in order to provide a specific system.
- the assay utilizing these purified antibodies does not cross react with HCG, FSH, or LH at normal levels of these hormones, improving the specificity of the assay for TSH.
- the FIGURE shows a standard curve.
- the assay method of this invention can be applied to the assay of TSH in serum.
- the reagents for a single-incubation two-site immunoradiometric assay for TSH are typically prepared as follows:
- the antibodies to TSH are produced by injection of TSH into rabbits, according to usual procedures. They are purified according to the principles of affinity chromatography, above.
- the tracer is prepared by iodinating purified antibody with the isotope 125 I to a specific activity of about 1 to 100 ⁇ Ci/ ⁇ g, preferably about 5 to 60 ⁇ Ci/ ⁇ g. However, other specific activities may be used.
- the antibody may be iodinated by conventional methods described in the literature.
- the tracer may be lyophilized from a solution containing phosphate buffer and carrier protein.
- the hydrolysis of polyacrylamide beads and the coupling of antibody to these beads is essentially as described in U.S. Pat. No. 4,088,746, although the hydrolysis may also be carried out at room temperature.
- the antibody which is coupled is preferably a globulin cut of the antiserum, although whole serum can be used. Usually from 0.25 to 2.0 ml of antibody is coupled to each gram of beads, preferably 0.75 to 1.5 ml per gram.
- the washing of the coupled beads is accomplished by use of phosphate buffer and 1M ammonium thiocyanate.
- the beads may be lyophilized from a solution containing phosphate buffer, protein, and HCG, although they can also be left in liquid suspension.
- the solid-phase receptor is used at a concentration such that under the assay conditions, the precipitation of TSH is essentially complete.
- the amount used per assay tube is approximately 1.0 to 5.0 mg, although other amounts may be used.
- the standards contain human TSH in human serum-based solution.
- the serum is stripped of endogenous TSH by charcoal and filtration prior to use.
- the protein environment of serum may be simulated by the use of dissolved serum or other proteins from human or animal origin.
- preservatives are added to the serum, conveniently sodium azide at 0.1% by weight.
- the human TSH contained in the standards are calibrated against the international reference preparation of human TSH supplied by the World Health Organization (Medical Research Council, Holly Hill, London). However, other standardizations may be used.
- the standards are lyophilized and they may be prepared to contain 0, 2.5, 5.0, 10, 25, 50 and 100 ⁇ IU/ml TSH, although other levels could be used.
- reaction parameters are typically as described below:
- Reaction volumes are kept reasonably small, although larger volumes can be used and the concentrations adjusted accordingly.
- 50 ⁇ l of tracer with about 50,000 to 300,000 dpm, preferably 100,000 to 200,000 dpm, is added to each assay tube.
- the volume of solid-phase receptor added to each tube may vary from about 100 ⁇ l to 500 ⁇ l, preferably 150 ⁇ l to 400 ⁇ l.
- the tracer and solid-phase receptor are preferably mixed and a single addition step is employed, but they may be added in two steps with the solid-phase receptor added first.
- the incubation temperature is preferably in the range of 2°-50° C., preferably 35°-40° C., the incubation time is conveniently as specified in Example 1, although times of from about one hour to about three hours could be used and acceptable standard curves obtained.
- mixing of the tubes is conveniently done with a laboratory Vortex mixer, although the tubes could also be mixed by manual agitation.
- the tracer is conveniently supplied in lyophilized form, although liquid tracer could also be used and appears to give similar stability characteristics.
- the solid-phase receptor is conveniently bottled and supplied as a suspension, although lyophilized preparations of solid-phase receptor appear to work equally well.
- the amounts of tracer and solid-phase receptor in each vial are adjusted so that prior to the assay, one vial of tracer could be reconstituted and mixed with one vial of solid-phase receptor.
- the number of tubes in a particular assay could be determined, and the appropriate amounts of tracer and solid-phase receptor could be mixed in a separate vial prior to the assay.
- the standards are conveniently supplied in lyophilized form.
- Each reagent must be thoroughly dissolved and mixed with the added water before use. All reagents and specimens should be allowed to come to room temperature before use.
- the tracer-bead reagent will be a fine suspension of polymeric particles and will appear cloudy. If more than 50 tubes are being run at one time, two vials of tracer should be reconstituted and mixed with two vials of solid-phase receptor in one vial, prior to use in the assay.
- This step comprises the single incubation mode or step of this invention.
- the concentrations of human thyroid stimulating hormone in the patient's samples and commercial control serum are determined from a standard curve.
- Standard curves may be obtained by several methods, for example, by plotting cpm vs. concentration.
- FIG. 1 shows a sample standard curve where cpm is plotted vs. concentration.
- the standard curve must be constructed for each assay, as the actual numbers will vary with the age of the reagents.
- kits for assaying a specimen for a multivalent ligand having improved reagents permitting the use of a single incubation mode as discussed above.
- kits will comprise containers of: (a) labeled receptor for said multivalent ligand; and (b) an unlabeled receptor for said multivalent ligand covalently bound to a solid-phase support which forms a substantially stable aqueous suspension.
- the kits will also comprise containers of standards for the multivalent ligand to be assayed.
- the kits in general may also comprise containers having the various other reagents previously described including those utilized in the preferred embodiment.
Abstract
This invention provides a two-site immunoassay method for a ligand utilizing a single incubation. The multivalent ligand, labeled receptor for the ligand and unlabeled receptor for the ligand covalently bound to a solid-phase support are incubated as a substantially stable suspension to produce a solid and liquid phase. The solid and liquid phases are separated from each other and the labeled receptor in either phase is quantitated.
The method may be used to assay for human thyroid stimulating hormone using purified, radioactively labeled antibodies and unlabeled antibodies covalently bound to hydrolyzed polyacrylamide particles.
Description
1. Field of the Invention
This invention relates to a novel method for a two-site immunoassay.
2. Description of the Prior Art
Two-site immunoradiometric assays for antigens which can bind to at least two antibodies are known. Those known two-site immunoradiometric assays involve a first incubation of unlabeled antibody coupled to an insoluble matrix with the fluid containing the antigen having more than one antigenic determinant, followed by a second incubation with a labeled antibody. Woodhead, Addison and Hales, Br. Med. Bull., Vol. 39, No. 1, 44-49 (1974); Abraham (ed.), Handbook of Radioimmunoassay, Ch. 4 (Marcel Dekker, Inc., 1977).
Such two-site immunoradiometric assays are discussed in Radioimmunoassay And Related Procedures In Medicine, Vol. 1, 131-47, 149-64 (International Atomic Energy Agency, Vienna 1974).
Corning Medical Diagnostics has marketed an assay for determination of human thyroid stimulating hormone (hereinafter referred to as "TSH"). See Corning, TSH (125 I) Radioimmunoassay Test System (March, 1977). In that assay as described by Corning, the radioactive, 125 I antibody, specific for TSH, is added to a known amount of TSH standard or to a patient serum sample. The radioactive, 125 I antibody will bind to any TSH present and form a labeled complex. After a first incubation of equilibration period, another antibody, also raised against TSH, is added to the reaction mixture. This antibody has been covalently bonded to microscopic glass particles and serves to bind the labeled complex formed in the first step. After binding separation can occur by simple centrifugation. The precipitated complex is counted for 125 I radioactivity. A standard curve is constructed in which radioactivity bound to glass is plotted against TSH concentration. The counts bound in the presence of unknown patient samples are compared to the standard curve and the appropriate TSH concentration is determined by interpolation.
Corning also markets an assay for the determination of human thyroxine-binding globulin ("TBG"). See Corning, TBG 125 I Radioimmunoassay Test System (December, 1977). In that assay, an immobilized antibody specific for TBG is added to a known amount of TBG standard or to a patient serum sample. After vortexing, radioactive 125 I labeled thyroxine ("125 I T4") is added to the reaction mixture. According to Corning, the 125 I T4 will then partition between the binding sites on TBG and bovine serum albumin molecules present in the buffer. The TBG can thus become bound both to the immobilized antibody and to the 125 I T4.
The use of antibodies covalently bound to a solid-phase in radioimmunoassays for proteins and polypeptides is shown by U.S. Pat. No. 3,555,143, issued Jan. 12, 1971, to Axen and Wide. Pharmacia has applied that method in a commercial radioimmunoassay for TSH. In this procedure, the antibody is covalently bound to a polydextran. The sample to be determined and the solid-phase antibody are incubated at room temperature for 18-24 hours followed by addition of radioactively labeled TSH and further incubation with agitation for 18-24 hours. The solid phase is separated, washed three times and its radioactivity measured.
The patent application by Monthony et al. Ser. No. 621,197, filed Oct. 9, 1975, describes an immunofluorescent assay method in which the immune reactants are covalently bound to water insoluble hydrophilic polymeric particles of about 0.1-10 microns in size. The solid phase antibody is mixed with the sample containing antigen (or hapten) to be determined and corresponding antigen (or hapten) which has been fluorescently labeled, so as to bind a quantity of labeled and unlabeled antigen. The solid phase is separated and the fluorescence measured by optical spectroscopy, the concentration of the unknown immune reactant being a function of the value of the fluorescence. This application also describes a two-site immunoassay method in which an excess of solid phase antibody is first reacted with the sample containing antigen (or hapten) to be determined, and the complex is subsequently reacted with an excess of fluorescently labeled antibody.
The present invention in an improved two-site immunoassay method in which the sample containing the multivalent ligand to be determined, a labeled receptor for said ligand, and an unlabeled receptor for said ligand covalently bound to a solid-phase support (hereinafter referred to as "solid-phase receptor") are brought together in an aqueous medium to form a substantially stable suspension and in a single incubation mode or step to produce a two-phase system, wherein the solid phase contains the solid-phase receptor, a portion of which has become bound to ligand which in turn has become bound to a portion of the labeled receptor, and the liquid phase contains the unbound portion of the labeled receptor. The solid and liquid phases are separated and either phase analyzed for the labeled receptor, the concentration of which is a function of the concentration of ligand in the sample.
A two-site immunoassay employing a single incubation mode or step provides a significant advantage over assay procedures involving more than one incubation by simplifying, shortening, and rendering more convenient the performance of the assay. This improvement in assay procedure is accomplished while maintaining acceptable assay characteristics such as precision, specificity, and stability, and in addition, is less subject to errors in timing, additions, and other manipulations.
The novel two-site immunoassay of this invention can be applied to any ligand which can simultaneously become bound by two receptors. This group of ligands includes, but is not limited to placental, pituitary, calcium regulating, and adrenal medullary polypeptide hormones, protein and protein fragments; immunoglobulins (antibodies) of various classes; viral, bacterial, and protozoal organisms or particles; enzymes; and tumor-associated antigens. In a preferred example, an assay for human thyroid stimulating hormone is described.
This assay method utilizes a receptor for the ligand, which is any substance which binds the ligand with acceptable specificity and affinity. Accordingly, the receptor may be, for example, an antibody against the ligand. In a preferred example, antibodies are used as the receptor.
The labeled receptor may be labeled with any of a number of known tracers, including, for example, radioactive tags, such as iodine 125 , or fluorescent labels. In a preferred example, radioactively labeled antibodies are employed.
An important component of this assay is the solid-phase support for unlabeled receptor which can form substantially stable suspensions in aqueous media, can be covalently bound to the unlabeled receptor, can be handled conveniently during manipulations such as pipetting and centrifuging, and exhibits low non-specific adsorption properties, or can be treated so that it exhibits such adsorption properties. The suspendability of the solid-phase support on which the unlabeled receptor is immobilized allows for the addition of an excess of solid-phase receptor, which binds the ligand in the sample, and yet permits further participation of the solid phase receptor-ligand complex in reactions with the labeled receptor. A suitable solid-phase support is derivatized polyacrylamide particles in a size range of 0.10 to 10 microns, which provides a substantially stable suspension during the single incubation period.
A preferred embodiment of this two-site immunoassay having a single incubation mode provides high specificity for TSH by using highly purified antibodies. The hormones TSH, HCG, FSH, and LH are each composed of similar alpha and distinct beta polypeptide chains. The hormones FSH, LH, and HCG may interfere with the assay for TSH due to the cross reactivity of the antibodies to those hormones. Improved specificity for TSH of this assay is achieved by a two step purification procedure for the labeled antibody and, if needed, purification of the unlabeled antibody to obtain antibodies of high affinity for TSH and low affinity for other hormones, for example HCG. Prepurification of receptor by these means constitutes an approach in dealing with inherent cross-reactivity due to common antigenic determinants in various subclasses of ligands and constitutes an improvement over prior art. In addition, prepurification of antibody makes it possible to introduce 125 I into a higher proportion of specific antibody molecules (subclass of the IgG fraction), and thus more of the radioactive label is usable in the assay.
The purification procedure for antibody to be labeled involves known principles of affinity chromatography to an immunoglobulin cut of rabbit anti-serum to TSH. In a two-step procedure, the immunoglobulins are first passed over an HCG column to remove anti-alpha chain antibodies, and then over a TSH column. The specific anti-beta TSH antibodies bind to the TSH column, and they are eluted off in stages so that the final antibody for labeling consists of essentially pure anti-beta TSH antiboidies of high affinity. In addition to purification of the labeled antibody, the unlabeled antibody may be evaluated for TSH specificity and, if necessary, either passed over an HCG column prior to coupling to the solid phase support or absorbed with HCG after coupling to the solid phase support in order to provide a specific system. The assay utilizing these purified antibodies does not cross react with HCG, FSH, or LH at normal levels of these hormones, improving the specificity of the assay for TSH.
The FIGURE shows a standard curve.
The assay method of this invention can be applied to the assay of TSH in serum.
The reagents for a single-incubation two-site immunoradiometric assay for TSH are typically prepared as follows:
The antibodies to TSH are produced by injection of TSH into rabbits, according to usual procedures. They are purified according to the principles of affinity chromatography, above. The tracer is prepared by iodinating purified antibody with the isotope 125 I to a specific activity of about 1 to 100 μCi/μg, preferably about 5 to 60 μCi/μg. However, other specific activities may be used. The antibody may be iodinated by conventional methods described in the literature. The tracer may be lyophilized from a solution containing phosphate buffer and carrier protein.
The hydrolysis of polyacrylamide beads and the coupling of antibody to these beads is essentially as described in U.S. Pat. No. 4,088,746, although the hydrolysis may also be carried out at room temperature. The antibody which is coupled is preferably a globulin cut of the antiserum, although whole serum can be used. Usually from 0.25 to 2.0 ml of antibody is coupled to each gram of beads, preferably 0.75 to 1.5 ml per gram. The washing of the coupled beads is accomplished by use of phosphate buffer and 1M ammonium thiocyanate. The beads may be lyophilized from a solution containing phosphate buffer, protein, and HCG, although they can also be left in liquid suspension.
The solid-phase receptor is used at a concentration such that under the assay conditions, the precipitation of TSH is essentially complete. The amount used per assay tube is approximately 1.0 to 5.0 mg, although other amounts may be used.
The standards contain human TSH in human serum-based solution. The serum is stripped of endogenous TSH by charcoal and filtration prior to use. Alternatively, the protein environment of serum may be simulated by the use of dissolved serum or other proteins from human or animal origin. Preferably preservatives are added to the serum, conveniently sodium azide at 0.1% by weight. The human TSH contained in the standards are calibrated against the international reference preparation of human TSH supplied by the World Health Organization (Medical Research Council, Holly Hill, London). However, other standardizations may be used. Preferably the standards are lyophilized and they may be prepared to contain 0, 2.5, 5.0, 10, 25, 50 and 100 μIU/ml TSH, although other levels could be used.
The reaction parameters are typically as described below:
Reaction volumes are kept reasonably small, although larger volumes can be used and the concentrations adjusted accordingly. Typically, 50 μl of tracer with about 50,000 to 300,000 dpm, preferably 100,000 to 200,000 dpm, is added to each assay tube. The volume of solid-phase receptor added to each tube may vary from about 100 μl to 500 μl, preferably 150 μl to 400 μl. The tracer and solid-phase receptor are preferably mixed and a single addition step is employed, but they may be added in two steps with the solid-phase receptor added first.
The incubation temperature is preferably in the range of 2°-50° C., preferably 35°-40° C., the incubation time is conveniently as specified in Example 1, although times of from about one hour to about three hours could be used and acceptable standard curves obtained. Prior to incubation, mixing of the tubes is conveniently done with a laboratory Vortex mixer, although the tubes could also be mixed by manual agitation.
The tracer is conveniently supplied in lyophilized form, although liquid tracer could also be used and appears to give similar stability characteristics. The solid-phase receptor is conveniently bottled and supplied as a suspension, although lyophilized preparations of solid-phase receptor appear to work equally well. The amounts of tracer and solid-phase receptor in each vial are adjusted so that prior to the assay, one vial of tracer could be reconstituted and mixed with one vial of solid-phase receptor. Alternatively, the number of tubes in a particular assay could be determined, and the appropriate amounts of tracer and solid-phase receptor could be mixed in a separate vial prior to the assay. The standards are conveniently supplied in lyophilized form.
Approximately thirty minutes before the assay is to be run:
1. Reconstitute 125 I-antibody to human TSH (tracer) with 2.5 ml distilled water.
2. Reconstitute Zero Standard with 5.0 ml distilled water.
3. Reconstitute standards containing 2.5-100 μIU/ml human TSH with 2.0 ml distilled water.
4. Prepare a normal saline solution (0.9% or 0.154 molar sodium chloride) in distilled water and store at room temperature.
5. Prepare tracer-bead reagent by combining one vial of 125 I-antibody with one vial of solid-phase receptor.
Each reagent must be thoroughly dissolved and mixed with the added water before use. All reagents and specimens should be allowed to come to room temperature before use. The tracer-bead reagent will be a fine suspension of polymeric particles and will appear cloudy. If more than 50 tubes are being run at one time, two vials of tracer should be reconstituted and mixed with two vials of solid-phase receptor in one vial, prior to use in the assay.
1. Label sixteen tubes in duplicate as follows: TC, zero, 2.5, 5.0, 10, 25, 50, and 100 μIU/ml. Label two tubes in duplicate for each patient serum and sample and for each commercial control serum.
2. Add 200 μl Standards zero through 100 to the appropriate tubes.
3. Add 200 μl of each patient's serum or commercial control serum to the appropriate tubes.
4. Mix tracer-bead reagent thoroughly and add 200 μl to all tubes. Set aside TC tubes.
5. Vortex tubes and incubate for two hours at 37° C. (except TC tubes). This step comprises the single incubation mode or step of this invention.
6. Add 3.0 ml saline to all tubes (except TC) and centrifuge for 10 minutes at 1500×g. Immediately after the centrifuge has stopped, decant tubes and blot the tube against filter paper or plastic backed absorbent paper.
7. Count all tubes (including TC) for one minute.
The concentrations of human thyroid stimulating hormone in the patient's samples and commercial control serum are determined from a standard curve. Standard curves may be obtained by several methods, for example, by plotting cpm vs. concentration. FIG. 1 shows a sample standard curve where cpm is plotted vs. concentration. The standard curve must be constructed for each assay, as the actual numbers will vary with the age of the reagents.
Sample data generated using this assay are shown below. These numbers were used to plot the standard curve in FIG. 1.
______________________________________ Sample cpm TSH Value ______________________________________ TC 144062,143313 Zero 6094,5464 0 μIU/ml 2.5 μIU/ml Standard 9318,9116 2.5 μIU/ml 5.0 μIU/ml Standard 11859,11745 5.0 μIU/ml 10 μIU/ml Standard 17992,15301 10 μIU/ml 25 μIU/ml Standard 25203,25241 25 μIU/ml 50 μIU/ml Standard 37266,35871 50 μIU/ml 100 μIU/ml Standard 49535,48363 100 μIU/ml Commercial Control I 13187,12766 6.2 μ IU/ml Commercial Control II 19773,19413 14.9 μIU/ml Commercial Control III 24519,25028 23.5 μIU/ml Patient 1 7009,6139 0.8 μIU/ml Patient 2 23466,24102 21.6 μIU/ml Patient 3 8373,7694 1.4 μIU/ml ______________________________________
The major use of the present invention from a practical standpoint takes place in clinical laboratories. Typically such laboratories obtain commercial kits containing the various reagents required to perform the assay. Accordingly, the present invention contemplates kits for assaying a specimen for a multivalent ligand having improved reagents permitting the use of a single incubation mode as discussed above. Such kits will comprise containers of: (a) labeled receptor for said multivalent ligand; and (b) an unlabeled receptor for said multivalent ligand covalently bound to a solid-phase support which forms a substantially stable aqueous suspension. Usually the kits will also comprise containers of standards for the multivalent ligand to be assayed. The kits in general may also comprise containers having the various other reagents previously described including those utilized in the preferred embodiment.
Claims (20)
1. An improved two-site immunoassay method, which comprises:
bringing together in an aqueous medium in an essentially single-incubation mode (a) the sample containing the multivalent ligand to be determined, (b) a labeled receptor for said ligand, and (c) an excess of unlabeled receptor to bind substantially all said ligand, said unlabeled receptor being covalently bound to a solid-phase support which forms a substantially stable suspension;
incubating said aqueous medium to produce a two-phase system, wherein the solid phase contains substantially all said ligand bound to both said unlabeled receptor and said labeled receptor, and the liquid phase contains the unbound portion of said labeled receptor;
separating said solid and liquid phases from each other; and
analyzing either phase for the labeled receptor, being a function of the concentration of said ligand in said sample.
2. An improved two-site immunoassay method in accordance with claim 1, wherein said solid-phase support is derivatized polyacrylamide particles of about 0.1 to 10 microns in size.
3. An improved two-site immunoassay method in accordance with claim 1, wherein said ligand is human thyroid stimulating hormone and said receptors for said ligand are antibodies against human thyroid stimulating hormone.
4. An improved two-site immunoassay in accordance with claim 3, wherein said labeled antibody against human thyroid stimulating hormone has been prepurified and consists essentially of those species of antibody against thyroid stimulating hormone having relatively high affinity and specificity for human thyroid stimulating hormone.
5. An improved two-site immunoassay method in accordance with claim 3, wherein said labeled antibody against said human thyroid stimulating hormone is labeled with a tractor atom or molecule.
6. An improved two-site immunoassay method in accordance with claim 5, wherein said tracer is a radioactive atom or molecule.
7. An improved two-site immunoassay method in accordance with claim 6, wherein said tracer is radioactive iodine 125.
8. An improved two-site immunoradiometric assay method for human thyroid stimulating hormone which comprises:
bringing together in an aqueous medium in essentially a single-incubation mode (a) the sample containing said human thyroid stimulating hormone to be determined, (b) a radioactively labeled antibody against human thyroid stimulating hormone, and (c) an excess of said unlabeled antibody to bind substantially all said human thyroid stimulating hormone, said unlabeled antibody being covalently bound to derivatized polyacrylamide particles of about 0.1 to 10 microns in size which forms a substantially stable suspension;
incubating said aqueous medium to produce a two-phase system, wherein the solid phase contains substantially all said hormone bound to both said unlabeled antibody and said labeled antibody, and the liquid phase contains the unbound portion of said labeled antibody;
separating said solid and liquid phases from each other; and
analyzing either phase for said labeled antibody, being a function of the concentration of sais hormone in said sample.
9. An improved two-site immunoradiometric assay according to claim 8, wherein said labeled antibody has been prepurified and consists essentially of those species of antibody having relatively high affinity and specificity for human thyroid stimulating hormone.
10. An improved two-site immunoradiometric assay according to claim 8, wherein said labeled antibody is labeled with radioactive iodine 125.
11. In an assay kit for assaying a specimen for a multivalent ligand, improved reagents permitting the use of a single incubation mode in the assay comprising containers of: (a) a labeled receptor for said multivalent ligand; and (b) an excess of unlabeled receptor to bind substantially all said multivalent ligand, said unlabeled receptor covalently bound to a solid-phase support which forms a substantially stable aqueous suspension.
12. The improved assay kit in accordance with claim 11 wherein said kit includes containers of standards for the multivalent ligand to be assayed.
13. The improved assay kit in accordance with claim 11 wherein said solid-phase support is derivatized polyacrylamide particles of about 0.1 to 10 microns in size.
14. The improved assay kit in accordance with claim 13 wherein said ligand is human thyroid stimulating hormone and said receptors for said ligand are antibodies against human thyroid stimulating hormone.
15. The improved assay kit in accordance with claim 14 wherein said labeled antibody against human thyroid stimulating hormone has been prepurified and consists essentially of those species of antibody against thyroid stimulating hormone having relatively high affinity and specificity for human thyroid stimulating hormone.
16. The improved assay kit in accordance with claim 14 wherein said labeled antibody against said human thyroid stimulating hormone is labeled with a tracer atom or molecule.
17. The improved assay kit in accordance with claim 16 wherein said tracer is a radioactive atom or molecule.
18. The improved assay kit in accordance with claim 17 wherein said tracer is radioactive iodine 125.
19. An improved two-site immunoassay method in accordance with claim 1, further comprising mixing together said excess of unlabeled receptor and said labeled receptor prior to said step of bringing together.
20. An improved two-site immunoradiometric assay method in accordance with claim 8, further comprising mixing together said excess of unlabeled antibody and said radioactively labeled antibody prior to said step of bringing together.
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US05/939,577 US4244940A (en) | 1978-09-05 | 1978-09-05 | Single-incubation two-site immunoassay |
GB7917063A GB2029571B (en) | 1978-09-05 | 1979-05-16 | Single-incubation two-site immunoassay |
DE19792925565 DE2925565A1 (en) | 1978-09-05 | 1979-06-25 | INCUBATIONAL DOUBLE SPACE IMMUNE DETECTION METHOD AND MEANS FOR IMPLEMENTING THE METHOD |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US05/939,577 US4244940A (en) | 1978-09-05 | 1978-09-05 | Single-incubation two-site immunoassay |
Publications (1)
Publication Number | Publication Date |
---|---|
US4244940A true US4244940A (en) | 1981-01-13 |
Family
ID=25473400
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US05/939,577 Expired - Lifetime US4244940A (en) | 1978-09-05 | 1978-09-05 | Single-incubation two-site immunoassay |
Country Status (3)
Country | Link |
---|---|
US (1) | US4244940A (en) |
DE (1) | DE2925565A1 (en) |
GB (1) | GB2029571B (en) |
Cited By (64)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS57148254A (en) * | 1981-02-13 | 1982-09-13 | Becton Dickinson Co | Analysis of antigen having multibonded parts and kit used therefor |
JPS57179750A (en) * | 1981-04-13 | 1982-11-05 | Hoechst Co American | Single culture immunity chemical examining method |
JPS5923251A (en) * | 1982-07-05 | 1984-02-06 | ベ−リンガ−・マンハイム・ゲゼルシヤフト・ミツト・ベシユレンクテル・ハフツング | Method of measuring polyvalent antigen and measuring reagent |
EP0105714A1 (en) * | 1982-09-29 | 1984-04-18 | Serono Diagnostics Limited | Immunoassay of antigens |
WO1984003358A1 (en) * | 1983-02-25 | 1984-08-30 | Covalent Technology Corp | Insoluble surfaces treated to inhibit non-specific protein binding |
US4471058A (en) * | 1982-07-26 | 1984-09-11 | Board Of Trustees Operating Michigan State University | Method for the detection and/or determination of a polyvalent antigen using at least two different monoclonal antibodies |
US4481298A (en) * | 1981-04-13 | 1984-11-06 | Amf Incorporated | Pre-precipitated double antibody immunoassay method |
WO1984004598A1 (en) * | 1983-05-06 | 1984-11-22 | Univ Columbia | Immunoassay for human chorionic gonadotropin |
US4486530A (en) * | 1980-08-04 | 1984-12-04 | Hybritech Incorporated | Immunometric assays using monoclonal antibodies |
US4496658A (en) * | 1980-10-15 | 1985-01-29 | Takeda Chemical Industries, Ltd. | Method for enzyme immunoassay and production of antibody |
US4514507A (en) * | 1980-11-07 | 1985-04-30 | Secher David S | Assay for interferon |
US4514505A (en) * | 1982-05-21 | 1985-04-30 | The Trustees Of Columbia University In The City Of New York | Monoclonal antibody mixtures and use thereof for enhanced sensitivity immunoassays |
US4522922A (en) * | 1982-04-16 | 1985-06-11 | Jose Carro | Soluble assay |
WO1986000992A1 (en) * | 1984-07-30 | 1986-02-13 | Shah Vipin D | Two site enzyme labeled cross reaction immunometric sandwich assay method |
US4596771A (en) * | 1982-09-27 | 1986-06-24 | Research Corporation | Monoclonal antibodies to vitamin B-6 and immunossay method |
US4652533A (en) * | 1983-04-28 | 1987-03-24 | Pandex Laboratories, Inc. | Method of solid phase immunoassay incorporating a luminescent label |
US4792528A (en) * | 1982-05-21 | 1988-12-20 | The Trustees Of Columbia University In The City Of New York | Methods for obtaining monoclonal antibodies useful in enhanced sensitivity immunoassays |
JPS64469A (en) * | 1987-03-03 | 1989-01-05 | Chugai Pharmaceut Co Ltd | Method and kit for measuring high-molecular hyaluronic acid |
US4837167A (en) * | 1981-01-30 | 1989-06-06 | Centocor, Inc. | Immunoassay for multi-determinant antigens using high-affinity |
US4889816A (en) * | 1980-06-20 | 1989-12-26 | Unilever Patent Holdings B.V. | Process and apparatus for carrying out specific binding assays |
US4935339A (en) * | 1985-05-07 | 1990-06-19 | Nichols Institute Diagnostics | Delayed solid phase immunologic assay |
US5009997A (en) * | 1980-06-30 | 1991-04-23 | Shah Vipin D | Two site cross-reaction immunometric sandwich assay method |
US5009996A (en) * | 1980-06-30 | 1991-04-23 | International Immunoassay Laboratories, Inc. | Two site cross-reaction immunometric sandwich assay method |
US5047207A (en) * | 1986-05-20 | 1991-09-10 | Agri-Diagnostics Associates | Kit for diagnosing plant diseases |
US5137804A (en) * | 1988-05-10 | 1992-08-11 | E. I. Du Pont De Nemours And Company | Assay device and immunoassay |
USRE34405E (en) * | 1983-08-01 | 1993-10-12 | Abbott Laboratories | Determination of analytes in particle-containing medium |
US5374524A (en) * | 1988-05-10 | 1994-12-20 | E. I. Du Pont De Nemours And Company | Solution sandwich hybridization, capture and detection of amplified nucleic acids |
US5391479A (en) * | 1992-10-29 | 1995-02-21 | E. I. Du Pont De Nemours And Company | Method for determining total analyte concentration in a sample having both free and bound analyte |
US5422239A (en) * | 1980-09-19 | 1995-06-06 | General Hospital Corporation | Immunoassay utilizing monoclonal high affinity IgM antibodies |
US5434051A (en) * | 1991-07-26 | 1995-07-18 | E. I. Du Pont De Nemours And Company | Assay with signal detection in the presence of a suspended solid support |
US5449556A (en) * | 1988-08-01 | 1995-09-12 | Ciba Corning Diagnostics Corp. | Method for detection of an analyte using acridinium esters and liposomes |
US5472849A (en) * | 1991-05-03 | 1995-12-05 | Boehringer Ingelheim Pharmaceuticals, Inc. | Method for detecting inflammation |
US5538901A (en) * | 1988-09-26 | 1996-07-23 | Ciba Corning Diagnostics Corp. | Nucleophilic polysubstituted aryl acridinium ester conjugates uses thereof |
US5602040A (en) * | 1987-04-27 | 1997-02-11 | Unilever Patent Holdings B.V. | Assays |
US5622871A (en) * | 1987-04-27 | 1997-04-22 | Unilever Patent Holdings B.V. | Capillary immunoassay and device therefor comprising mobilizable particulate labelled reagents |
US5663074A (en) * | 1988-09-26 | 1997-09-02 | Chiron Diagnostics Corporation | Nucleophilic polysubstituted aryl acridinium ester conjugates and syntheses thereof |
US5726015A (en) * | 1991-05-15 | 1998-03-10 | Vanderbilt University | Method to determine metastatic potential of tumor cells |
US5739042A (en) * | 1993-12-23 | 1998-04-14 | Sinvent As | Method of assay |
US5929049A (en) * | 1997-08-08 | 1999-07-27 | Dade Behring Marburg Gmbh | Polysaccharide conjugates of biomolecules |
US6153442A (en) * | 1998-05-20 | 2000-11-28 | Dade Behring Inc. | Reagents and methods for specific binding assays |
US6201109B1 (en) | 1993-01-13 | 2001-03-13 | Dade Behring Marburg Gmbh | Assay for bone alkaline phosphatase |
US6352862B1 (en) | 1989-02-17 | 2002-03-05 | Unilever Patent Holdings B.V. | Analytical test device for imuno assays and methods of using same |
US6406920B1 (en) | 1980-06-20 | 2002-06-18 | Inverness Medical Switzerland Gmbh | Processes and apparatus for carrying out specific binding assays |
KR20020065699A (en) * | 2001-02-07 | 2002-08-14 | 주식회사 바이오라인 | Detection kit of thyroid stimulating hormone using immunoradiometric assays |
US6449562B1 (en) | 1996-10-10 | 2002-09-10 | Luminex Corporation | Multiplexed analysis of clinical specimens apparatus and method |
US6524793B1 (en) | 1995-10-11 | 2003-02-25 | Luminex Corporation | Multiplexed analysis of clinical specimens apparatus and method |
US20040014781A1 (en) * | 2000-05-31 | 2004-01-22 | Walter Elger | Compounds with sulphonamide group and pharmaceutical compositions containing these compounds |
US6844162B1 (en) * | 1998-06-06 | 2005-01-18 | Rsr Limited | Assays for TSH receptor autoantibodies |
US20050164261A1 (en) * | 2001-10-09 | 2005-07-28 | Chandler Don J. | Multiplexed analysis of clinical specimens apparatus and methods |
US20050214882A1 (en) * | 2004-03-25 | 2005-09-29 | Ez Bio Inc. | Reagents, methods and kits for the universal rapid immuno-detection |
US20050277625A1 (en) * | 2004-05-21 | 2005-12-15 | Ralf Wyrwa | Estriol and estetrol prodrugs |
US20050288267A1 (en) * | 2004-05-21 | 2005-12-29 | Ralf Wyrwa | Estradiol prodrugs |
US20070123500A1 (en) * | 2005-11-29 | 2007-05-31 | Gerd Mueller | Prodrugs of ERbeta-selective substances, processes for their preparation and pharmaceutical compositions comprising these compounds |
US20070135399A1 (en) * | 2005-11-30 | 2007-06-14 | Ralf Wyrwa | Heteroaromatic sulphonamide prodrugs |
US20070135375A1 (en) * | 2005-11-30 | 2007-06-14 | Ralf Wyrwa | Sulfamoyl sulfonate prodrugs |
US20070148640A1 (en) * | 2000-09-25 | 2007-06-28 | Scopp Richard L | Methods and kits for decreasing interferences in plasma or serum containing assay samples of specific binding assays |
US20070197488A1 (en) * | 2005-11-29 | 2007-08-23 | Olaf Peters | Prodrugs of ERbeta-selective substances, process for their production, and pharmaceutical compositions that contain these compounds |
US20090203036A1 (en) * | 2001-08-23 | 2009-08-13 | Rsr Limited | Epitope regions of a thyrotrophin (tsh) receptor, uses thereof and antibodies thereto |
US8148171B2 (en) | 2001-10-09 | 2012-04-03 | Luminex Corporation | Multiplexed analysis of clinical specimens apparatus and methods |
JP2012242170A (en) * | 2011-05-17 | 2012-12-10 | Tosoh Corp | Measurement container |
WO2016141270A3 (en) * | 2015-03-04 | 2016-10-27 | Sanaria Inc. | Serum antibody assay for determining protection from malaria, and pre-erythrocytic subunit vaccines |
US9739773B1 (en) | 2010-08-13 | 2017-08-22 | David Gordon Bermudes | Compositions and methods for determining successful immunization by one or more vaccines |
WO2019211218A1 (en) | 2018-05-02 | 2019-11-07 | Hahn-Schickard-Gesellschaft Für Angewandte Forschung E. V. | Immunoassay for an automated system |
CN113777297A (en) * | 2021-08-14 | 2021-12-10 | 浙江大学 | Fluorescence differential rapid detection method based on magnetic nanoparticles |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA1160566A (en) * | 1980-04-25 | 1984-01-17 | Harald Gallati | Immunological determination method |
EP0042755B1 (en) * | 1980-06-20 | 1988-08-24 | Unilever Plc | Processes and apparatus for carrying out specific binding assays |
NL185309C (en) * | 1980-07-28 | 1990-03-01 | Akzo Nv | METHOD FOR DETERMINING ANTIGENS USING TWO OR MORE MONOCLONAL ANTIBODIES AND IMMUNE REAGENTS. |
JPS58500037A (en) * | 1980-11-07 | 1983-01-06 | セルテク リミテツド | Interferon assay method |
JPS57136165A (en) * | 1981-02-18 | 1982-08-23 | Mochida Pharmaceut Co Ltd | Immunological measuring reagent |
AU649725B2 (en) * | 1990-10-22 | 1994-06-02 | Sumitomo Electric Industries, Ltd. | Assay of substances using flow cytometry |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3555143A (en) * | 1966-06-02 | 1971-01-12 | Pharmacia Ab | Method for the determination of proteins and polypeptides |
US3853987A (en) * | 1971-09-01 | 1974-12-10 | W Dreyer | Immunological reagent and radioimmuno assay |
US3995019A (en) * | 1975-03-04 | 1976-11-30 | Baxter Travenol Laboratories, Inc. | Diagnostic reagent system |
US4017597A (en) * | 1974-10-30 | 1977-04-12 | Monsanto Company | Unitized solid phase immunoassay kit and method |
US4034073A (en) * | 1975-03-28 | 1977-07-05 | Corning Glass Works | Composite for biased solid phase radioimmunoassay of triiodothyronine and thyroxine |
US4088746A (en) * | 1976-11-11 | 1978-05-09 | Bio-Rad Laboratories, Inc. | Radioimmunoassay for thyroid-stimulating hormone (TSH) |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB1572220A (en) * | 1976-10-07 | 1980-07-30 | Mochida Pharm Co Ltd | Immunochemical process of measuring physiologically active substances |
-
1978
- 1978-09-05 US US05/939,577 patent/US4244940A/en not_active Expired - Lifetime
-
1979
- 1979-05-16 GB GB7917063A patent/GB2029571B/en not_active Expired
- 1979-06-25 DE DE19792925565 patent/DE2925565A1/en not_active Withdrawn
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3555143A (en) * | 1966-06-02 | 1971-01-12 | Pharmacia Ab | Method for the determination of proteins and polypeptides |
US3853987A (en) * | 1971-09-01 | 1974-12-10 | W Dreyer | Immunological reagent and radioimmuno assay |
US4017597A (en) * | 1974-10-30 | 1977-04-12 | Monsanto Company | Unitized solid phase immunoassay kit and method |
US3995019A (en) * | 1975-03-04 | 1976-11-30 | Baxter Travenol Laboratories, Inc. | Diagnostic reagent system |
US4034073A (en) * | 1975-03-28 | 1977-07-05 | Corning Glass Works | Composite for biased solid phase radioimmunoassay of triiodothyronine and thyroxine |
US4088746A (en) * | 1976-11-11 | 1978-05-09 | Bio-Rad Laboratories, Inc. | Radioimmunoassay for thyroid-stimulating hormone (TSH) |
Non-Patent Citations (3)
Title |
---|
Abraham, Ed, Handbook of Radioimmuno Assay, Marcel Dekker, Inc., N.Y., 1977, pp. 131-134. * |
Radioimmuno Assay and Related Procedures in Medicine, International Atomic Energy Agency, Vienna, 1974, pp. 131-147, 149-164. * |
Woodhead et al., Br. Med. Bull., vol. 30, No. 1, 1974, pp. 44-49. * |
Cited By (95)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6228660B1 (en) | 1908-04-27 | 2001-05-08 | Conopco Inc. | Capillary immunoassay and device therefor comprising mobilizable particulate labelled reagents |
US5610077A (en) * | 1980-06-20 | 1997-03-11 | Unilever Patent Holdings B.V. | Processes and apparatus for carrying out specific binding assays |
US6406920B1 (en) | 1980-06-20 | 2002-06-18 | Inverness Medical Switzerland Gmbh | Processes and apparatus for carrying out specific binding assays |
US4889816A (en) * | 1980-06-20 | 1989-12-26 | Unilever Patent Holdings B.V. | Process and apparatus for carrying out specific binding assays |
US5009996A (en) * | 1980-06-30 | 1991-04-23 | International Immunoassay Laboratories, Inc. | Two site cross-reaction immunometric sandwich assay method |
US5009997A (en) * | 1980-06-30 | 1991-04-23 | Shah Vipin D | Two site cross-reaction immunometric sandwich assay method |
US4486530A (en) * | 1980-08-04 | 1984-12-04 | Hybritech Incorporated | Immunometric assays using monoclonal antibodies |
US5422239A (en) * | 1980-09-19 | 1995-06-06 | General Hospital Corporation | Immunoassay utilizing monoclonal high affinity IgM antibodies |
US4496658A (en) * | 1980-10-15 | 1985-01-29 | Takeda Chemical Industries, Ltd. | Method for enzyme immunoassay and production of antibody |
US4514507A (en) * | 1980-11-07 | 1985-04-30 | Secher David S | Assay for interferon |
US4837167A (en) * | 1981-01-30 | 1989-06-06 | Centocor, Inc. | Immunoassay for multi-determinant antigens using high-affinity |
JPS57148254A (en) * | 1981-02-13 | 1982-09-13 | Becton Dickinson Co | Analysis of antigen having multibonded parts and kit used therefor |
JPS57179750A (en) * | 1981-04-13 | 1982-11-05 | Hoechst Co American | Single culture immunity chemical examining method |
US4481298A (en) * | 1981-04-13 | 1984-11-06 | Amf Incorporated | Pre-precipitated double antibody immunoassay method |
US4522922A (en) * | 1982-04-16 | 1985-06-11 | Jose Carro | Soluble assay |
US4514505A (en) * | 1982-05-21 | 1985-04-30 | The Trustees Of Columbia University In The City Of New York | Monoclonal antibody mixtures and use thereof for enhanced sensitivity immunoassays |
US4792528A (en) * | 1982-05-21 | 1988-12-20 | The Trustees Of Columbia University In The City Of New York | Methods for obtaining monoclonal antibodies useful in enhanced sensitivity immunoassays |
JPS5923251A (en) * | 1982-07-05 | 1984-02-06 | ベ−リンガ−・マンハイム・ゲゼルシヤフト・ミツト・ベシユレンクテル・ハフツング | Method of measuring polyvalent antigen and measuring reagent |
JPH0413659B2 (en) * | 1982-07-05 | 1992-03-10 | Boehringer Mannheim Gmbh | |
US4471058A (en) * | 1982-07-26 | 1984-09-11 | Board Of Trustees Operating Michigan State University | Method for the detection and/or determination of a polyvalent antigen using at least two different monoclonal antibodies |
US4596771A (en) * | 1982-09-27 | 1986-06-24 | Research Corporation | Monoclonal antibodies to vitamin B-6 and immunossay method |
EP0105714A1 (en) * | 1982-09-29 | 1984-04-18 | Serono Diagnostics Limited | Immunoassay of antigens |
WO1984003358A1 (en) * | 1983-02-25 | 1984-08-30 | Covalent Technology Corp | Insoluble surfaces treated to inhibit non-specific protein binding |
US4652533A (en) * | 1983-04-28 | 1987-03-24 | Pandex Laboratories, Inc. | Method of solid phase immunoassay incorporating a luminescent label |
WO1984004598A1 (en) * | 1983-05-06 | 1984-11-22 | Univ Columbia | Immunoassay for human chorionic gonadotropin |
USRE34405E (en) * | 1983-08-01 | 1993-10-12 | Abbott Laboratories | Determination of analytes in particle-containing medium |
WO1986000992A1 (en) * | 1984-07-30 | 1986-02-13 | Shah Vipin D | Two site enzyme labeled cross reaction immunometric sandwich assay method |
US4935339A (en) * | 1985-05-07 | 1990-06-19 | Nichols Institute Diagnostics | Delayed solid phase immunologic assay |
US5047207A (en) * | 1986-05-20 | 1991-09-10 | Agri-Diagnostics Associates | Kit for diagnosing plant diseases |
JPS64469A (en) * | 1987-03-03 | 1989-01-05 | Chugai Pharmaceut Co Ltd | Method and kit for measuring high-molecular hyaluronic acid |
US7109042B2 (en) | 1987-04-27 | 2006-09-19 | Inverness Medical Switzerland Gmbh | Assays |
US6818455B2 (en) | 1987-04-27 | 2004-11-16 | Inverness Medical Switzerland Gmbh | Capillary immunoassay and device therefor comprising mobilizable particulate labelled reagents |
US6187598B1 (en) | 1987-04-27 | 2001-02-13 | Conopco Inc. | Capillary immunoassay and device therefor comprising mobilizable particulate labelled reagents |
US5602040A (en) * | 1987-04-27 | 1997-02-11 | Unilever Patent Holdings B.V. | Assays |
US5656503A (en) * | 1987-04-27 | 1997-08-12 | Unilever Patent Holdings B.V. | Test device for detecting analytes in biological samples |
US5622871A (en) * | 1987-04-27 | 1997-04-22 | Unilever Patent Holdings B.V. | Capillary immunoassay and device therefor comprising mobilizable particulate labelled reagents |
US5391478A (en) * | 1988-05-10 | 1995-02-21 | E. I. Du Pont De Nemours And Company | Assay device and immunoassay |
US5137804A (en) * | 1988-05-10 | 1992-08-11 | E. I. Du Pont De Nemours And Company | Assay device and immunoassay |
US5374524A (en) * | 1988-05-10 | 1994-12-20 | E. I. Du Pont De Nemours And Company | Solution sandwich hybridization, capture and detection of amplified nucleic acids |
US5595875A (en) * | 1988-08-01 | 1997-01-21 | Ciba Corning Diagnostics Corp. | Method for detection of an analyte using acridinium ester and liposomes |
US5656500A (en) * | 1988-08-01 | 1997-08-12 | Chiron Diagnostics Corporation | Luminescent conjugates for use in luminescent assays |
US5449556A (en) * | 1988-08-01 | 1995-09-12 | Ciba Corning Diagnostics Corp. | Method for detection of an analyte using acridinium esters and liposomes |
US5663074A (en) * | 1988-09-26 | 1997-09-02 | Chiron Diagnostics Corporation | Nucleophilic polysubstituted aryl acridinium ester conjugates and syntheses thereof |
US5538901A (en) * | 1988-09-26 | 1996-07-23 | Ciba Corning Diagnostics Corp. | Nucleophilic polysubstituted aryl acridinium ester conjugates uses thereof |
US6080591A (en) * | 1988-09-26 | 2000-06-27 | Bayer Corporation | Nucleophilic polysubstituted aryl acridinium ester conjugates and syntheses thereof |
US7384796B2 (en) | 1989-02-17 | 2008-06-10 | Inverness Medical Switzerland Gmbh | Assays |
US7407813B2 (en) | 1989-02-17 | 2008-08-05 | Inverness Medical Switzerland Gmbh | Assays |
US20030219908A1 (en) * | 1989-02-17 | 2003-11-27 | Davis Paul James | Assays |
US20030143755A1 (en) * | 1989-02-17 | 2003-07-31 | Davis Paul James | Assays |
US7238537B2 (en) | 1989-02-17 | 2007-07-03 | Inverness Medical Switzerland Gmbh | Assays |
US6352862B1 (en) | 1989-02-17 | 2002-03-05 | Unilever Patent Holdings B.V. | Analytical test device for imuno assays and methods of using same |
US5472849A (en) * | 1991-05-03 | 1995-12-05 | Boehringer Ingelheim Pharmaceuticals, Inc. | Method for detecting inflammation |
US5726015A (en) * | 1991-05-15 | 1998-03-10 | Vanderbilt University | Method to determine metastatic potential of tumor cells |
US5434051A (en) * | 1991-07-26 | 1995-07-18 | E. I. Du Pont De Nemours And Company | Assay with signal detection in the presence of a suspended solid support |
US5654159A (en) * | 1991-07-26 | 1997-08-05 | Dade International Inc. | Assay with signal detection in the presence of a suspended solid support |
US5391479A (en) * | 1992-10-29 | 1995-02-21 | E. I. Du Pont De Nemours And Company | Method for determining total analyte concentration in a sample having both free and bound analyte |
US6201109B1 (en) | 1993-01-13 | 2001-03-13 | Dade Behring Marburg Gmbh | Assay for bone alkaline phosphatase |
US5739042A (en) * | 1993-12-23 | 1998-04-14 | Sinvent As | Method of assay |
US20050118574A1 (en) * | 1995-10-11 | 2005-06-02 | Luminex Corporation | Multiplexed analysis of clinical specimens apparatus and method |
US6524793B1 (en) | 1995-10-11 | 2003-02-25 | Luminex Corporation | Multiplexed analysis of clinical specimens apparatus and method |
US6939720B2 (en) | 1995-10-11 | 2005-09-06 | Luminex Corporation | Multiplexed analysis of clinical specimens apparatus and method |
US6449562B1 (en) | 1996-10-10 | 2002-09-10 | Luminex Corporation | Multiplexed analysis of clinical specimens apparatus and method |
US5929049A (en) * | 1997-08-08 | 1999-07-27 | Dade Behring Marburg Gmbh | Polysaccharide conjugates of biomolecules |
US6949524B2 (en) | 1997-08-08 | 2005-09-27 | Dade Behring Marburg Gmbh | Polysaccharide conjugates of biomolecules |
US20050281833A1 (en) * | 1997-08-08 | 2005-12-22 | Dade Behring Marburg Gmbh | Polysaccharide conjugates of biomolecules |
US6489309B1 (en) | 1997-08-08 | 2002-12-03 | Dade Behring Marburg Gmbh | Polysaccharide conjugates of biomolecules |
US6153442A (en) * | 1998-05-20 | 2000-11-28 | Dade Behring Inc. | Reagents and methods for specific binding assays |
US6844162B1 (en) * | 1998-06-06 | 2005-01-18 | Rsr Limited | Assays for TSH receptor autoantibodies |
US20040014781A1 (en) * | 2000-05-31 | 2004-01-22 | Walter Elger | Compounds with sulphonamide group and pharmaceutical compositions containing these compounds |
US20070148640A1 (en) * | 2000-09-25 | 2007-06-28 | Scopp Richard L | Methods and kits for decreasing interferences in plasma or serum containing assay samples of specific binding assays |
US20110136256A1 (en) * | 2000-09-25 | 2011-06-09 | Abbott Laboratories | Methods and kits for decreasing interferences in plasma or serum containing assay samples of specific binding assays |
KR20020065699A (en) * | 2001-02-07 | 2002-08-14 | 주식회사 바이오라인 | Detection kit of thyroid stimulating hormone using immunoradiometric assays |
US9751940B2 (en) | 2001-08-23 | 2017-09-05 | Rsr Limited | Epitope regions of a thyrotrophin (TSH) receptor, uses thereof and antibodies thereto |
US8298771B2 (en) | 2001-08-23 | 2012-10-30 | Rsr Limited | Epitope regions of a thyrotrophin (TSH) receptor, uses thereof and antibodies thereto |
US8298769B2 (en) | 2001-08-23 | 2012-10-30 | Rsr Limited | Epitope regions of a thyrotrophin (TSH) receptor, uses thereof and antibodies thereto |
US8309693B2 (en) | 2001-08-23 | 2012-11-13 | Rsr Limited | Epitope regions of a thyrotrophin (TSH) receptor, uses thereof and antibodies thereto |
US20090203036A1 (en) * | 2001-08-23 | 2009-08-13 | Rsr Limited | Epitope regions of a thyrotrophin (tsh) receptor, uses thereof and antibodies thereto |
US8148171B2 (en) | 2001-10-09 | 2012-04-03 | Luminex Corporation | Multiplexed analysis of clinical specimens apparatus and methods |
US20050164261A1 (en) * | 2001-10-09 | 2005-07-28 | Chandler Don J. | Multiplexed analysis of clinical specimens apparatus and methods |
CN101421619B (en) * | 2004-03-25 | 2012-06-06 | 邹松 | Reagents, methods and kits for the universal rapid immuno-detection |
US20050214882A1 (en) * | 2004-03-25 | 2005-09-29 | Ez Bio Inc. | Reagents, methods and kits for the universal rapid immuno-detection |
US20100041076A1 (en) * | 2004-03-25 | 2010-02-18 | Ez Bio Inc. | Reagents, Methods and Kits for the Universal Rapid Immuno-Detection |
US7534780B2 (en) | 2004-05-21 | 2009-05-19 | Bayer Schering Pharma Aktiengesellschaft | Estradiol prodrugs |
US20050288267A1 (en) * | 2004-05-21 | 2005-12-29 | Ralf Wyrwa | Estradiol prodrugs |
US20050277625A1 (en) * | 2004-05-21 | 2005-12-15 | Ralf Wyrwa | Estriol and estetrol prodrugs |
US20070197488A1 (en) * | 2005-11-29 | 2007-08-23 | Olaf Peters | Prodrugs of ERbeta-selective substances, process for their production, and pharmaceutical compositions that contain these compounds |
US20070123500A1 (en) * | 2005-11-29 | 2007-05-31 | Gerd Mueller | Prodrugs of ERbeta-selective substances, processes for their preparation and pharmaceutical compositions comprising these compounds |
US20070135375A1 (en) * | 2005-11-30 | 2007-06-14 | Ralf Wyrwa | Sulfamoyl sulfonate prodrugs |
US20070135399A1 (en) * | 2005-11-30 | 2007-06-14 | Ralf Wyrwa | Heteroaromatic sulphonamide prodrugs |
US9739773B1 (en) | 2010-08-13 | 2017-08-22 | David Gordon Bermudes | Compositions and methods for determining successful immunization by one or more vaccines |
JP2012242170A (en) * | 2011-05-17 | 2012-12-10 | Tosoh Corp | Measurement container |
WO2016141270A3 (en) * | 2015-03-04 | 2016-10-27 | Sanaria Inc. | Serum antibody assay for determining protection from malaria, and pre-erythrocytic subunit vaccines |
US10197577B2 (en) | 2015-03-04 | 2019-02-05 | Sanaria Inc. | Serum antibody assay for determining protection from malaria, and pre-erythrocytic subunit vaccines |
WO2019211218A1 (en) | 2018-05-02 | 2019-11-07 | Hahn-Schickard-Gesellschaft Für Angewandte Forschung E. V. | Immunoassay for an automated system |
CN113777297A (en) * | 2021-08-14 | 2021-12-10 | 浙江大学 | Fluorescence differential rapid detection method based on magnetic nanoparticles |
Also Published As
Publication number | Publication date |
---|---|
GB2029571A (en) | 1980-03-19 |
DE2925565A1 (en) | 1980-03-13 |
GB2029571B (en) | 1983-02-16 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US4244940A (en) | Single-incubation two-site immunoassay | |
EP0162914B1 (en) | Novel immunoassays and kits for use therein | |
CA1226215A (en) | Immunoassay of antigens | |
US4551426A (en) | Heterogeneous immunoassay for digoxin using ouabain as a separation means | |
US4378344A (en) | Method and apparatus for performing multiple, simultaneous in vitro diagnostic tests using a solid phase system | |
US4092408A (en) | Method for solid phase immunological assay of antigen | |
US3985867A (en) | Immunoassays employing a colored second antibody | |
EP0105714A1 (en) | Immunoassay of antigens | |
WO1983004312A1 (en) | Monoclonal antibody mixtures and use thereof for enhanced sensitivity immunoassays | |
IE48400B1 (en) | Process for immunologic determination tests | |
US4088746A (en) | Radioimmunoassay for thyroid-stimulating hormone (TSH) | |
WO1985000663A1 (en) | Immunometric assay using polyclonal and monoclonal antibodies and a kit for use therein | |
US5366859A (en) | Radioimmunoassay method | |
Haber et al. | Radio immunoassay employing gel filtration | |
US4522922A (en) | Soluble assay | |
EP0088974A2 (en) | Homogeneous immunoassay with labelled monoclonal anti-analyte | |
FI78787B (en) | IMMUNOMETRISK METHOD FOR THE END OF HAPPEN. | |
GB1597556A (en) | Device for use in immunoassays | |
US5196349A (en) | Immunoassays for thyroid hormones using thyroglobulin | |
US4820633A (en) | Process for production of an immune reactive, porous carrier material for heterogeneous immunological analysis | |
JP3502497B2 (en) | Competitive immunoassay using conjugated analyte derivatives | |
Jeong et al. | Single-incubation immunoassay for a multivalent ligand | |
Parker | [53] Immunoassays | |
US4312854A (en) | Immunoassay for thyroid stimulating hormone employing florisil adsorbent as separating agent | |
Sutherland et al. | Immunoassay of blood spot TSH; development of a rapid two-site immunoradiometric assay and comparison with radioimmunoassay as a screening method for neonatal hypothyroidism |